Extracellular Matrix Materials as Vaccine Adjuvants for Diseases Associated with Infectious Pathogens or Toxins

ABSTRACT

Disclosed are vaccines and vaccine adjuvants useful in the treatment and/or prevention of infection and diseases associated with infectious pathogens, such as tetanus, as well as diseases associated with biological toxins. Also provided are methods of preparing an adjuvant and the vaccine containing the adjuvant. Methods are also provided for vaccinating/immunizing an animal against infection and diseases associated with infectious pathogens, such as tetanus, and other diseases associated with biological toxins. Adjuvant materials are presented that are prepared from an extracellular matrix material. The adjuvants are demonstrated to enhance the immunogenicity of an infectious pathogen antigen or biological toxin antigen of interest, as well as to enhance the survival of an immunized animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/699,448 filed Jan. 30, 2007, which is a Continuation-in-part of U.S.patent application Ser. No. 11/583,771, filed Oct. 20, 2006, whichclaims priority from U.S. Provisional Patent Application No. 60/730,379,filed Oct. 27, 2005. The entire disclosure and contents of the aboveapplications are hereby incorporated by reference.

STATEMENT OF JOINT RESEARCH AGREEMENT

In compliance with 37 C.F.R. §1.71(g)(1), disclosure is herein made thatthe claimed invention was made pursuant to a Joint Research Agreement asdefined in 35 U.S.C. 103(c)(3), that was in effect on or before the datethe claimed invention was made, and as a result of activities undertakenwithin the scope of the Joint Research Agreement, by or on the behalf ofthe University of Notre Dame and Cook Biotech, Inc. (West Lafayette,Ind.).

BACKGROUND

1. Field of the Invention

The present invention relates generally to vaccines that include anadjuvant, and to adjuvants alone. In particular, the invention relatesto adjuvants derived or obtained at least in part from biologicaltissues, such as extracellular matrices, particularly small intestinaltissue (SIS). The invention also relates to the field of methods forimmunizing an animal against diseases associated with infectiouspathogens, and infections by said pathogens, or toxins using a vaccinepreparation that includes a tissue-derived adjuvant. The invention alsorelates to the field of methods for preparing adjuvants, as a method forpreparing an adjuvant from small intestinal tissue for use as a part ofa vaccine to immunize an animal against diseases associated with aninfectious agent, and in particular, against tetanus, as a vaccine forthe treatment and/or prevention of tetanus, is provided.

2. Related Art

Aluminum hydroxide and aluminum phosphate (collectively referred to asalum) are routinely used as adjuvants in human and veterinary vaccines(1). The efficacy of alum in increasing antibody responses to diphtheriaand tetanus toxins is well established (2) and Hepatitis B virus antigenvaccine has been adjuvinated with alum (3). While the usefulness of alumis well established for some applications, it has limitations. Forexample, alum is a poor inducer of Th1 cellular immune responses andstimulates the production of antibodies, which is consistent with Th2cellular immune response (4-6). Unfortunately, a Th2 based immuneresponse is not likely to offer optimal protection against severalimportant infectious diseases, including tuberculosis (TB), humanimmunodeficiency virus (HIV) and hepatitis C virus (HCV). Alum is poorlyeffective for influenza vaccination and inconsistently elicits a cellmediated immune response. The antibodies elicited by alum-adjuvinatedantigens are mainly of the IgG1 isotope in the mouse, which may beoptimal for protection by some vaccinal agents.

Tetanus is an important human and animal disease characterized bypainful, uncontrolled muscle spasms, and death due to paralysis of therespiratory muscles. This disease is associated with infection byClostridium tetani and prophylactic vaccination is common. Tetanusvaccines typically use alum as an adjuvant.

A need continues to exist in the medical arts for materials that may beused to enhance and/or improve existing clinical alternatives to thetreatment and prophylaxis of disease associated with infectious agentsand toxins, for example, to improve existing forms of tetanus treatmentvaccines and tetanus vaccine adjuvants with improved immunogenicity.

SUMMARY OF THE INVENTION

The present invention was developed in part by the inventors'recognition of the robust inflammatory response invoked by anextracellular matrix material (ECM) preparation, such as matrix isolatedfrom the small intestinal submucosa (SIS). While not intending to belimited to any particular mechanism of action, the extracellular matrixmaterial appears to provide the robust inflammatory response through,among other things, its contribution of pro-inflammatory species thatdrive the immune response to the antigenic species that it isco-administered with. The present invention harnesses theinflammatory-provoking activity of ECM, such as SIS, and preparationsfrom other forms of ECM, in the design of an immunopotent infectiousagent vaccine preparation and infectious agent adjuvant.

The crafting of infectious agent vaccine preparations using ECM, andmaterials like it, may be used in combination with many differentinfectious pathogens or biological toxins. By way of example, and insome embodiments, the biological toxin is tetanus toxin. By further wayof example, and in some embodiments, the biological toxin is ricin.

The present invention is unique in that, among other things, it involvesthe modification and use of a three-dimensional extracellular matrixmaterial, and modified preparations thereof, to provide a vaccine. Byway of example, and in some embodiments, the vaccine is a tetanusvaccine. The invention thus provides in some embodiments highly improvedinfectious agent preparations with an adjuvant material having anacceptable biocompatibility.

The adjuvant effect of the ECM, such as the SIS adjuvant preparation,extends to a vaccine administered to protect against diseases associatedwith an infectious pathogen or biological toxin. In some embodiments,the present invention provides an adjuvant comprising an SIS gel orparticulate SIS. Administered together with tetanus toxoid, thesepreparations confer protective immunity in vivo to animals challengedwith tetanus toxin.

Infectious Agent Adjuvant

In one aspect, the present invention provides an extracellular matrix(ECM) material, such as a modified preparation of SIS, as an infectiousagent vaccine adjuvant. In some embodiments, these preparations may bedescribed as essentially free of alum. In some embodiments, the ECMmaterials may be described as a modified preparation of SIS (diluted)about 2-fold to about 20-fold.

Infectious Agent Vaccine

In another aspect, the present invention provides an infectious agentvaccine comprising a preparation of an extracellular matrix materialtogether with a preparation of an antigen of an infectious pathogen.

In one aspect of the invention, there is provided an adjuvantcomposition comprising an immunogenically enhancing preparationcharacteristic of an extracellular matrix material (ECM), particularly apreparation comprising an extracellular matrix derived from smallintestinal mucosa (SIS) or renal capsule material (RCM). In particularembodiments, the adjuvant composition comprises an extracellular matrixmaterial comprising a small intestinal mucosa tissue preparation. Insome embodiments, the adjuvant composition comprises 1 part of anextracellular matrix material (ECM) and 9 parts of a pharmaceuticallyacceptable carrier solution. By way of example, such a carrier issterile saline.

According to another aspect, there is provided a composition comprisingan adjuvant and a antigen of interest. In some embodiments, the antigenis a toxoid antigen. In some embodiments, the vaccine may be describedas a vaccine to protect against infectious pathogens, such as a tetanusvaccine, an influenza vaccine, a rabies vaccine, a viral hepatitisvaccine, a diphtheria vaccine, an anthrax vaccine, a Streptococcuspneumonia infection vaccine, a malaria vaccine, a leishmaniasis vaccine,or a Staphylococcal enterotoxin B toxicosis vaccine.

Biological Toxins

Examples of the biological toxins that may be used in the preparation ofthe vaccines of the present invention are provided below:

Abrin Aflatoxins

Botulinum toxinsClostridium perfringens episilon toxin

Conotoxins Diacetoxyscirpenol Ricin Saxitoxin Shigatoxin

Staphylococcal enterotoxins

Tetrodotoxin T-2 Toxin

Diptheria toxinStreptococcal toxinsCholera toxinPertussis toxin

Pneumolysin

In particular embodiments, the vaccine may be described as a vaccine toprotect against diseases associated with biological toxins, such asricin.

Prion-Associated Diseases:

In some embodiments, the invention provides an adjuvant preparation thatis suitable for use in combination with a prion-associated disease. Byway of example, such prion associated diseases include, all of which areclassified as transmissible spongiform encephalopathies, bovinespongiform encephalopathy, scrapie, ceroid chronic wasting disease andCreutzfeld-Jakob disease.

In other embodiments, the invention may be described as providing avaccine to protect against disease associated with a viral infection. Byway of example, the vaccines of the present invention may be formulatedto provide a composition useful in the treatment and/or prevention ofviral infections associated with influenza, rabies and viral hepatitis.

In other embodiments, the invention may be described as providing avaccine to protect against diseases associated with a bacterialinfection. By way of example, the vaccines of the present invention maybe formulated to provide a composition useful in the treatment and/orprevention of bacterial infections associated with diseases such asdiphtheria, anthrax, sepsis, pneumonia, otitis media and meningitis.

In other embodiments, the invention may be described as providing avaccine to protect against diseases associated with a parasiticinfection. By way of example, the vaccines of the present invention maybe formulated to provide a composition useful in the treatment and/orprevention of a parasitic infection associated with the diseases ofmalaria and leishmaniasis.

In other embodiments, the invention may be described as providing avaccine to protect against illnesses and/or diseases associated withexposure to a biological toxin. By way of example, the vaccines of thepresent invention may be formulated to provide a composition useful inthe treatment and/or prevention of illness associated with exposure tobiological toxins such as ricin (that causes respiratory distress) orexposure to Staphylococcal enterotoxin B (SEB) (that results in foodpoisoning).

Method of Preparing a Adjuvant and a Vaccine

In another aspect, the invention provides a method for preparing aninfectious agent vaccine. In some embodiments, the method comprisespreparing an adjuvant for vaccination against infectious agents andbiological toxins as described herein, and combining the adjuvant withan immunizing antigen of interest. In some embodiments, the antigen ofinterest is a tetanus toxoid preparation.

Methods of Preventing, Treating, Inhibiting, and/or Immunizing an Animalagainst an Infectious Pathogen

According to yet another broad aspect of the invention, a method fortreating an animal having an infectious disease or at risk ofcontracting a disease or illness associated with exposure to aninfectious pathogen. By way of example, diseases associated withexposure to an infection pathogen include tetanus, malaria, diphtheria,anthrax, sepsis, pneumonia, otitis media and meningitis. In someembodiments, the invention provides a method for immunizing an animalagainst tetanus. In yet another embodiment, the invention provides amethod for inhibiting the severity of tetanus and/or preventing theonset of tetanus altogether.

Clinical Infectious Pathogen Treatment Preparations

In yet another aspect, the invention provides a variety of uniqueinfectious pathogen treatment preparations. These infectious agenttreatment preparations may take the form of a gel, a sheet, or aninjectable preparation suitable for parenteral administration, combinedwith an appropriate antigen of interest.

The following abbreviations are used throughout the description of thepresent invention:

ECM—Extracellular Matrix;

HCV—Hepatitis C Virus;

HIV—Human Immunodeficiency Virus

RCM—Renal Capsule Material

SIS—Small Intestinal Submucosa;

TB—Tuberculosis

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, according to some embodiments of the invention, presents aremnant of SIS extracellular matrix material in a rat 28 days aftersurgical implantation. The remaining biomaterial is surrounded bymacrophages with occasional lymphocytes. Stained with H & E, 400X.

FIG. 2, according to some embodiments of the invention, presents thefocus of mononuclear inflammation at the interface of an implant/tendonsurface in a rat which underwent repair of Achilles tendon defect withRCM 7 days earlier. Stained with H & E, 200X.

FIG. 3, according to some embodiments of the invention, presents thesurvival data of mice, vaccinated with 0.03 micrograms of tetanustoxoid, following challenge with 1 ng/mouse of tetanus toxinintraperitoneally. Treatment groups are untreated (None); SIS gel (SG);SIS particulate (SP); tetanus toxoid (TT); TT with alum (TT/Alum); TTwith SIS gel (TT/SG); and TT with SIS particulate (TT/SP). Each groupconsisted of 15 mice. All mice which were untreated or vaccinated withonly SIS gel or SIS particulate died; six mice vaccinated withunadjuvanted tetanus toxoid survived, and all mice vaccinated withtetanus toxoid in alum, SIS gel, or SIS particulate survived. Thisrepresents a significant (P<0.001) increase in number of mice survivingfor the latter three groups compared to all other groups.

FIG. 4, according to some embodiments of the invention, presentssurvival data of mice, vaccinated with 0.05 micrograms of tetanustoxoid, following challenge with 1 ng/mouse of tetanus toxinintraperitoneally. Treatment groups are untreated (None); SIS gel (SG);SIS particulate (SP); tetanus toxoid (TT); TT with alum (TT/Alum); TTwith SIS gel (TT/SG); and TT with SIS particulate (TT/SP). Each groupconsisted of 15 mice. All mice which were untreated or vaccinated withonly SIS gel or SIS particulate died; eight mice vaccinated withunadjuvanted tetanus toxoid survived; and all mice vaccinated withtetanus toxoid in alum, SIS gel, or SIS particulate survived. Thisrepresents a significant (P<0.001) increase in number of mice survivingfor the latter three groups compared to all other groups.

DETAILED DESCRIPTION

It is advantageous to define several terms before describing theinvention. It should be appreciated that the following definitions areused throughout this application.

Definitions

Where the definition of terms departs from the commonly used meaning ofthe term, applicant intends to utilize the definitions provided below,unless specifically indicated.

For the purposes of the present invention, the term “adjuvant” isdefined as a substance which enhances the immune response to an antigen.

For purposes of the present invention, the term, “adjuvancy” is definedas the ability of an agent to enhance and/or promote the immune responseof animal to a particular antigen.

For the purposes of the present invention, the term “biosyntheticmaterial” is defined as a material that is in part or whole made up fromor derived from a biological tissue.

For purposes of the present invention, the term “biological tissue” isdefined as an animal tissue, including human, or plant tissue that is orthat once was (cadaver tissue, for example) part of a living tissue ororganism.

For the purposes of the present invention, the term “extracellularmatrix” is defined as a tissue derived or bio-synthetic material that iscapable of supporting the growth of a cell or culture of cells.

For the purposes of the present invention, the term “infectious agent”is defined as any bacterial, viral, prion or parasitic agent capable ofcausing disease in humans or animals subsequent to infection orsecretion of a substance, such as the production of a toxin or toxins.This term also includes the toxic products of such agents. By way ofexample, such an infectious agent includes clostridium botulinum, thecausative agent of tetanus.

For the purposes of the present invention, the term “biological toxin”is a poisonous substance, especially a protein that is produced byliving cells or organisms and is capable of causing disease whenintroduced into the body tissues, such as ricin or Staphylococcalenterotoxin B and tetanus toxin.

For the purposes of the present invention, the term, “immunogenicamount” is an amount of an infectious pathogen antigen preparation ofinterest or amount of a biological toxin that elicits a clinicallydetectable protective response in an animal. By way of example, aclinically detectable protective response in an animal may be theproduction of an elevated titer of antibodies in the animal specific forthe infectious pathogen antigen or biological toxin.

Description

A method for providing a preparation having an enhanced activity forinhibiting and protecting against an infectious pathogen provided. Inparticular embodiments, the infectious pathogen is tetanus.

A method for the treatment and/or inhibition of an infection caused byan infectious pathogen is also provided. In some embodiments, the methodemploys a composition comprising a vaccine, the vaccine comprising anadjuvant having a menu of pro-inflammatory species characteristic of anextracellular matrix (ECM) material together with an antigen associatedwith an infectious pathogen or a biological toxin. These preparationsare found to be more immunogenic than use of the infectious pathogenantigen or biological toxin antigen vaccine alone in the treatmentand/or prophylaxis against an infectious pathogen or biological toxin,such as tetanus.

The immune response to the described tetanus vaccine is enhanced by useof SIS as an adjuvant.

The description of the present invention is enhanced by the variousexamples that follow.

Example 1 Materials and Methods for ECM as an Adjuvant for a Vaccineagainst Diseases Associated with an Infectious Pathogen

The present example provides some examples of materials and methods thatmay be used in the practice of the present invention.

Small Intestinal Submucosa (SIS)

Small Intestinal Submucosa (SIS) was obtained from Cook Biotech, Inc.(West Lafayette, Ind.). Experimental grade material was provided for usein the present studies of an SIS preparation that was described ashaving been prepared by harvesting porcine jejunum and placing 10- to20-cm lengths into saline solution (31-33). Following removal of allmesenteric tissues, the jejunal segment was everted and the tunicamucosa abraded using a longitudinal wiping motion with a scalpel handleand moistened gauze. The serosa and tunica muscularis were then gentlyremoved using the same procedure. The remaining tissue was disinfectedwith peracetic acid, rinsed extensively in high purity water, andsterilized using ethylene oxide. SIS Particulate is supplied by CookBiotech, Inc. (West Lafayette, Ind.) and is SIS material ground andsieved. The size particles are in the range from 45 micron to 335micron. SIS gel is supplied by Cook Biotech, Inc. (West Lafayette, Ind.)and is produced from SIS material via an acid digestion and purificationprocess.

Tetanus Toxin and Tetanus Toxoid

Tetanus toxin and tetanus toxoid were purchased from List BiologicalLaboratories (Campbell, Calif.).

Alum

Alum was purchased as Alhydrogel™, an aluminum hydroxide gel adjuvant(Brenntak Biosector, Frederikssund, Denmark).

Animals (Mice)—Statistical Analysis

Results of survival versus non-survival following challenge with tetanustoxin were compared between groups using the Chi-square test with twodegrees of freedom. Differences were considered significant whenp≦0.001.

Example 2 Use of Adjuvant in the Inhibition of Tetanus

To determine if SIS can act as an adjuvant for vaccines against diseasesassociated with infectious pathogens or biological toxins, preparationswere made with tetanus toxoid, an inactivated form of tetanus toxin.Both gel SIS and SIS particles produced from a sheet of single layer SISwere evaluated as adjuvants. Briefly, groups of 15 Balb/C female mice(Harlan, Inc., Indianapolis, Ind.) were vaccinated initially (0.1 mlvolume/dose) and again, five weeks later with one of the following:

Particulate SIS

Gel SIS

Tetanus toxoid (TT; 0.03 ug/dose)

TT (0.05 ug/dose)

TT (0.03 ug/dose)+alum (alhydrogel)

TT (0.05 ug/dose)+alum (alhydrogel)

TT (0.03 ug/dose)+particulate SIS

TT (0.05 ug/dose)+particulate SIS

TT (0.03 ug/dose)+gel SIS

TT (0.05 ug/dose)+gel SIS

Untreated control group

Five weeks after the second vaccination, mice were challenged with alethal dose of tetanus toxin (1 ng/mouse) given intraperitoneally in 0.2ml of sterile saline. Mice were then observed over the next 96 hours andthe number of surviving mice recorded for each group.

The results of this study are as follows:

Treatment Group Number Surviving/Total at 96 h Untreated  0/15 Gel SIS 0/15 Particulate SIS  0/15 Tetanus toxoid (TT; 0.03 ug/dose)  6/15 TT(0.05 ug/dose)  8/15 TT (0.03 ug/dose) + alum (alhydrogel) 15/15 TT(0.05 ug/dose) + alum (alhydrogel) 15/15 TT (0.03 ug/dose) + particulateSIS 15/15 TT (0.05 ug/dose) + particulate SIS 15/15 TT (0.03 ug/dose) +gel SIS 15/15 TT (0.05 ug/dose) + gel SIS 15/15

A significantly greater number of mice survived challenge with tetanustoxin in groups vaccinated with either 0.03 or 0.05 μg/dose of tetanustoxoid administered in alhydrogel, SIS gel, or particulate SIS comparedto all other vaccination groups.

These results demonstrate the ability of both particulate and gel SIS toact as an adjuvant for a vaccine against disease associated with aninfectious pathogen, such as tetanus.

Example 3 Exemplary Infectious Pathogens

The present example demonstrates the utility of the present inventionwith disease associated with a wide variety of infectious pathogens andbiological toxins, including by way of example and not exclusion,tetanus, influenza, rabies, viral hepatitis, diphtheria, anthrax,Streptococcus pneumoniae infection, malaria, leishmaniasis, ricintoxicosis, and Staphylococcal enterotoxin B toxicosis.

TABLE 2 Classification of Common Vaccines for Humans Disease or PathogenType of Vaccine Whole Organisms: Bacterial cells: Cholera InactivatedPlague Inactivated Tuberculosis Attenuated BCG+ Salmonella typhiAttenuated Viral Particles: Influenza Inactivated Measles AttenuatedMumps Attenuated Rubella Attenuated Polio (Sabin/OPV) Attenuated Polio(Salk/IPV) Inactivated V. zoster Attenuated Yellow fever Attenuated Typeof Vaccine (Purified) Macromolecules Toxoids: Diphtheria Inactivatedexotoxin Tetanus Inactivated exotoxin acellular Pertussis Inactivatedexotoxins Capsular polysaccharide: Haemophilus influenzae bpolysaccharide + protein carrier Neisseria meningidis PolysaccarideStreptococcus pneumoniae 23 distinct capsular polysaccharides Surfaceantigen: Hepatitis B Recombinant surface antigen (HbsAg) +BacillusCalmette-Guerin (BCG) is an antiviral strain of Mycobacterium bovis.

Vaccines for Disease Associated with Viral Infections

1. Influenza—Influenza is an acute febrile respiratory disease resultingfrom infection with the influenza virus. Current influenza vaccines usealuminum adjuvants. To enhance the efficacy of vaccines, severaladjuvants have been examined. For example, the oil-in-water emulsionMF59 has been reported to improve vaccine immunity (Higgins (1996)¹;Martin (1997)), though it does not completely solve the low efficiencyof the influenza vaccine in the elderly (Banzhoff (2003)³). A syntheticpeptide, GKI, derived from Taenia crassiceps cysticerci was reported toenhance the immune response accompanying influenza vaccination in bothyoung and aged mice (Segura-Velasquez (2006)⁴), but trials in humanshave not been published.

As part of the present invention, an influenza vaccine may be providedthat comprises the extracellular matrix material described herein as thevaccine adjuvant combined with an immunologically effective amount of aninfluenza antigen. By way of example, such an influenza antigen maycomprise a current influenza virus combination of antigens of an H5NI(hemagglutinin [HA] subtype 1; neuraminidase [NA] subtype 1), and H3N2influenza A virus, and an influenza B virus. This preparation and otherinfluenza antigen preparations are described in Palese (2006)³³. Thisarticle and all of its teachings are incorporated herein by reference.

2. Rabies—Rabies is a devastating neurological disease that is caused byinfection with the rabies virus. Vaccination against rabies typicallyutilizes inactivated virus and an aluminum adjuvant. A lipoid adjuvantof the oil-in-water type, based on squalene, significantly increased theimmunologic response of mice to vaccination with an inactivated virusvaccine when compared to vaccination using an aluminum salt adjuvant(Suli, 2004). An adjuvant based on glycopeptidolipids extracted fromMycobacterium cheloniae enhanced the immune response of mice tovaccination with an inactivated rabies virus vaccine (de Souza Matos(2000)⁶).

As part of the present invention, a rabies vaccine may be provided thatcomprises the extracellular matrix material as the vaccine adjuvantcombined with an immunologically effective amount of a rabies antigen.By way of example, a rabies antigen may comprise an inactivated rabiesvirus. One example of an inactivated rabies virus vaccine antigen thatmay be used in the present formulations is described in de Souza Matos(2000)⁶.

3. Viral Hepatitis—Viral hepatitis, particularly that caused byHepatitis B virus, is a serious health problem with over 300 millionpeople affected worldwide. Vaccination offers hope for effectiveprophylaxis. Peptide epitopes of the virus stimulated a significantimmune response when fused with heat shock protein 70 from Mycobacteriumtuberculosis as an adjuvant (Peng (2006)⁷). Unmethylated CpGdinucleotides were effective as an adjuvant with hepatitis B antigen inaged mice (Qin (2004)⁸); and a vaccine consisting of hepatitis B virusantigens and an immunostimulatory DNA sequence is in human clinicaltrials (Sung (2006)⁹). In development of an intranasal vaccine, it wasshown that DL-lactide/glycolide copolymer microspheres with chitosanwere an effective adjuvant for a vaccine based on recombinant HepatitisB surface protein (Jaganathan (2006)¹⁰).

As part of the present invention, a viral hepatitis vaccine may beprovided that comprises the extracellular matrix material as the vaccineadjuvant combined with an immunologically effective amount of a viralhepatitis antigen. By way of example, such a hepatitis antigen maycomprise recombinant hepatitis B surface protein. By way of example,such a hepatitis B surface protein antigen is described in Jaganathan,(2006)¹⁰), which reference is specifically incorporated herein byreference.

Vaccines for disease associated with bacterial infections:

1. Diphtheria—A respiratory disease characterized by dysnepea, weakness,and pyrexia, diphtheria is the result of infection with Corynebacteriumdiphtheriae, bacteria which produces a toxin that is carriedhematogenously through the body. Immunization against diphtheria isfrequently combined with immunization against tetanus and pertussis;these vaccines typically contain aluminum salt adjuvants (Sugai(2005)¹¹). Unmethylated CpG dinucleotides were effective as an adjuvantin a diphtheria-tetanus-pertussis vaccine and shifted the immuneresponse toward cell-mediated immunity in mice immunizedintraperitoneally (Sugai (2005)¹¹). Trials to reduce adverseside-effects related to the aluminum salt adjuvant of a vaccineconsisting of diphtheria toxoid, tetanus toxoid, and purified Bordetellapertussis antigens including pertussis toxoid showed that reduction ofthe aluminum salt content of the vaccine resulted in reduced geometricmean antibody concentrations to the relevant antigens, but did notresult in reduction of local or general side effects (Theeten (2005)¹²).Monophosphoryl lipid A was shown in mice to effectively serve as anadjuvant for diphtheria toxin in mice (Caglar (2005)¹³).

As part of the present invention, a diphtheria vaccine may be providedthat comprises the extracellular matrix material as the vaccine adjuvantcombined with an immunologically effective amount of a diphtheriaantigen. By way of example, a diphtheria antigen may comprise adiphtheria toxoid. One example of a diphtheria toxoid that may be usedin the practice of the present invention is described in Theeten(2005)¹².

2. Anthrax—Anthrax is a disease caused by the bacterium, Bacillusanthracis. Specifically, the bacterium produces a toxin which results inhemorrhagic necrosis of lymph nodes, hematogenous spread, shock, anddeath. A vaccine consisting of one subunit (protective antigen) of thistoxin was shown to protect mice when combined with a microparticleadjuvant administered by either the intramuscular or intranasal routes(Flick-Smith (2002)¹⁴. Further, vaccination protected mice againstinfection with B. anthracis spores. While the aluminum salt-adjuvantedanthrax-vaccine-adsorbed is the only anthrax vaccine licensed in theUnited States, major drawbacks exist, including a very lengthy andcomplicated dosing schedule, followed by annual booster injections.Further, the aluminum adjuvant of anthrax vaccine has been linked toGulf War Illness among veterans of the 1991 conflict (Petrik (2007))¹⁵.

As part of the present invention, an anthrax vaccine may be providedthat comprises the extracellular matrix material as the vaccine adjuvantcombined with an immunologically effective amount of an anthrax antigen.By way of example, such an anthrax antigen may comprise the one subunit(protective antigen) of the Bacillus anthracis bacterium. One suchparticular antigenic subunit is described in Flick-Smith (2002)¹⁴.

3. Streptococcus pneumoniae—A bacterial pathogen of particularimportance to the elderly and young adults, Streptococcus pneumoniaecauses disease including sepsis and pneumonia, otitis media andmeningitis. Vaccines typically involve adsorption of S. pneumoniaeantigens to aluminum salt adjuvants, and reduced aluminum salt contentled to reduced immunogenicity of S. pneumoniae vaccines (Levesque(2006)¹⁶. In human trials, IL-12 failed to improve the immune responseto a pneumococcal polysaccharide vaccine; and IL-12 was associated witha high incidence of local and systemic side effects in humans (Hedlund(2002)¹⁷. Intranasal immunization against S. pneumoniae has been shownto be an effective method for preventing infection and disease, withunmethylated CpG dinucleotides serving as an effective adjuvant for anintranasal polysaccharide-protein conjugate vaccine (Sen (2006)¹⁸).Likewise, IL-12 and the B-subunit of cholera toxin were both shown toenhance efficacy of intranasally-administered preparations of S.pneumoniae antigens (Sabirov (2006)¹⁹; Pimenta (2006)²⁰).

As part of the present invention, a pneumonia vaccine may be providedthat comprises the extracellular matrix material described herein as thevaccine adjuvant combined with an immunologically effective amount of apneumococcal antigen. By way of example, such a pneumococcal antigen maycomprise a pneumococcal polysaccharide antigen. One form of apneumococcal polysaccharide antigen is described in Hedlund (2002)¹⁷.This pneumococcal antigen may used as part in combination with theherein described adjuvants in a vaccine preparation.

Vaccines for Diseases Associated with Parasitic Infections

1. Malaria—Malaria affects millions of people worldwide and each year,1-2 million people die from the disease caused by Plasmodium falciparum.Thus, the need for prophylactic measures has led to great interest inanti-malaria vaccines. The apical membrane antigen, a malaria vaccinecandidate, was reported to have an enhanced immunogenicity by thealuminum salt adjuvant Alhydrogel (HCI Biosector, Denmark); and thisadjuvant effect was further enhanced, and shifted from a Th1 response toa mixed Th1/Th2 response, by inclusion of the adjuvant CpGoligodeoxynucleotide (Mullen (2006)²¹). Alhydrogel and Montanide ISA 720(Seppic, France) were compared in rhesus monkeys as adjuvants for avaccine based on protective epitopes from the circumsporozoite proteinof P. falciparum. Though Montanide ISA 720 induced superior immuneresponses, the formation of sterile abscesses at injection sites werenoted as a significant disadvantage (Langermans (2005)²²). Other studieswith a circumsporozoite protein vaccine conducted in rhesus monkeysshowed that some novel oil-in-water adjuvants with components ofimmunostimulants 3-deacetylated monophosphoryl lipid A (3D-MPL) and thesaponin Quillaja saponaria 21 (QS21) were safe and stimulated improvedantibody responses (Stewart (2006)²³). Some of these same oil-in-wateradjuvants improved the immune response to a vaccine constructed of theP. falcipanim antigen, Liver Stage Antigen-1 (Brando (2006)²⁴).

As part of the present invention, a malarial vaccine may be providedthat comprises the extracellular matrix material as the vaccine adjuvantcombined with an immunologically effective amount of a malarial antigen.By way of example, such a malarial antigen may comprise a P. falciparumantigen Liver Stage Antigen-1. This antigen is described in detail inBrando (2006)²⁴, this article being specifically incorporated herein byreference. This antigen may be combined with the extracellular matrixmaterial described herein as an adjuvant to provide an anti-malarialvaccine as described herein.

2. Leishmaniasis—Leishmaniasis is a parasitic disease associated withinfection by a species of parasites from the Leishmania genus. A largespectrum of clinical disease forms can result from infection, rangingfrom cutaneous lesions to fatal visceral forms. In the absence ofeffective, non-toxic treatments, great effort has been given to vaccinedevelopment. Vaccines based on DNA of the parasite have been shown toinduce partial protection; aluminum phosphate adjuvant has no effect onthe humoral response to this vaccine, but has been reported to slightlyincrease the cellular immune response and protection against infectionin a mouse model (Rosado-Vallado (2005)²⁵). In evaluations in rhesusmonkeys using a soluble Leishmania antigen and alum with IL-12 asadjuvants, it was shown that the adjuvants improved protective immunity,though transient nodules developed at the site of subcutaneous injection(Kenney (1999)²⁶). CpG oligodeoxynucleotides served as an effectiveadjuvant for a vaccine consisting of live, nonattenuated L. majororganisms alone or in combination with lysates of heat-killed L. majorpromastigotes, either without or bound to alum (Mendez (2003)²⁷).Partial protective immunity was stimulated, but mice receivingalum-containing vaccines developed large dermal lesions that required upto 10 weeks to heal.

As part of the present invention, an anti-parasitic infection associateddisease vaccine may be provided that comprises the extracellular matrixmaterial as the vaccine adjuvant combined with an immunologicallyeffective amount of a Leishmaniasis antigen, or any of the otherantigenic species described above. By way of example, a Leishmaniasisantigen may comprise the Leishmaniasis antigen described in detail inKenny (1999)²⁶, which article is specifically incorporated herein byreference.

Vaccines for disease associated with biological toxins

1. Ricin—Ricin is a toxin produced naturally by the seeds of the castorbean plant, Ricinus communis. When humans or animals are exposed to thetoxin, severe respiratory distress and death may result. Because of itspotency and ability to be administered via aerosol, ingestion, orinjection, ricin is considered a powerful bioweapon. Though there ispresently no approved commercial vaccine for ricin, pilot trials inhumans have examined the use of recombinant, non-toxic forms of one ofthe subunits of ricin (Vitetta (2006)²⁸). This preparation wasadministered without an adjuvant and elicited ricin-neutralizingantibodies in some of those tested, particularly at higher doses.However, all dose groups were found to result in significantside-effects, including myalgia and headache. Ricin toxoid adjuvantizedby liposomal encapsulation was found to induce a stronger immuneresponse when administered intra-tracheally than the vaccineadjuvantized with an aluminum salt adjuvant (Griffiths (1997)²⁹). Avaccine consisting of a deglycosylated chain A ricin (DCAR) and theadjuvant LTR72, a mutant of the heat-labile enterotoxin of Escherichiacoli, resulted in a stronger antibody response of vaccinated mice toricin, but did not result in improved protection against lung injurywhen challenged with ricin (Kende)(2006)³⁰.

As part of the present invention, an anti-ricin vaccine may be providedthat comprises the extracellular matrix material as the vaccine adjuvantas described herein combined with an immunologically effective amount ofa ricin toxoid antigen. By way of example, such a ricin toxoid antigenis described in detail in Griffiths (1997)²⁹, which article isspecifically incorporated herein by reference.

2. Staphylococcal enterotoxin B (SEB)—SEB is produced by the bacteria,Staphylococcus aureus and is associated with food poisoning.Incorporation of SEB toxoid into biodegradablepoly(DL-lactide-co-glycolide) microspheres enhanced the immune responseof mice to a degree similar to SEB toxoid adsorbed to alum and combinedwith complete Freund adjuvant (Eldridge, 1991)³¹). Similarly, SEB toxoidwas effectively adjuvantized by incorporation into polylacticpolyglycolic acid copolymer nanospheres; the resulting immune responsewas comparable to that achieved by using alum as an adjuvant (Desai(2000)³²).

As part of the present invention, an anti-toxin-associated diseasevaccine may be provided that comprises the extracellular matrix materialas the vaccine adjuvant combined with an immunologically effectiveamount of an antigen such as ricin toxoid or SEB toxoid as antigen. Byway of example, such antigens are described in detail in Vitetta(2006)²⁸ and Eldridge (1991)³¹, the teachings of which are specificallyincorporated herein by reference.

Vaccines for Diseases Associated with Prions:

In some embodiments, the invention provides an adjuvant preparation thatis suitable for use in combination with a prion-associated disease. Byway of example, such prion associated diseases include, all of which areclassified as transmissible spongiform encephalopathies, bovinespongiform encephalopathy, scrapie, cervid chronic wasting disease andCreutzfeld-Jakob disease.

Although prions use immune and lymphoreticular cells to gain access tothe brain (Aguzzi, 2003)³⁶, existing evidence suggests that humoralimmune responses can suppress infection. In particular, antibodies tothe cellular prion protein (PrPc) are known to inhibit prion propagation(Petetz, 2001³⁷; Enari, 2001³⁸). Still, host tolerance to endogenousPrPc remains a major obstacle to active vaccination. In mice,vaccination with recombinant PrPc antigens such as peptides andpolypeptides stimulated only weak immune responses. Co-administration ofprion antigens with adjuvants such as Freund's (Polymenidou, 2004³⁹;Koller, 2002⁴⁰; Sigurddson, 2002⁴¹; Gilch, 2003⁴²; Hanan, 2001⁴³; Hanan,2001⁴⁴; Souan, 2001⁴⁵; Arbel, 2003⁴⁶); Montanide IMS-1313 (Schwartz,2003⁴⁷); TiterMax®, a combination of a proprietary block copolymerCRL-8941, squalene, a metabolizable oil, and a unique microparticulatestabilizer (Gilch, 2003⁴²); and CpG oligonucleotides (Rosset, 2004⁴⁸)all failed to induce strong immune responses.

It is anticipated that the presently described adjuvant preparations ofan extracellular matrix material may be used with the prion protein(PrPc) to provide an improved vaccine against prion-associatedinfections.

All documents, patents, journal articles and other materials cited inthe present application are hereby incorporated by reference.

Although the present invention has been fully described in conjunctionwith several embodiments thereof with reference to the accompanyingdrawings, it is to be understood that various changes and modificationsmay be apparent to those skilled in the art. Such changes andmodifications are to be understood as included within the scope of thepresent invention as defined by the appended claims, unless they departtherefrom.

BIBLIOGRAPHY

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide background for or teach methodology,techniques and/or compositions employed herein.

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1. A method for immunizing an animal against an infectious pathogen ofinterest comprising: administering to an animal an immunogenic amount ofa composition comprising an antigen of an infectious pathogen ofinterest and an adjuvant, wherein said adjuvant comprises anextracellular matrix and is essentially free of alum, to provide animmunized animal; and eliciting an immunologically detectable titer ofantibodies specific for the infectious pathogen in said animal.
 2. Themethod of claim 1 wherein the infectious pathogen antigen of interestcomprises a tetanus toxoid preparation.
 3. The method of claim 2 whereinthe immunogenic amount of the composition comprises 0.03 μg to 0.05 μgof the tetanus toxoid.
 4. The method of claim 1 wherein the immunizedanimal has an enhanced survival rate upon exposure to the infectiouspathogen of interest compared to an animal not receiving the
 5. Themethod of claim 1 wherein the composition comprises an antigen ofinterest for immunizing an animal against tetanus, influenza, rabies,viral hepatitis, diphtheria, anthrax, Streptococcus pneumoniaeinfection, malaria, leishmaniasis, ricin toxicosis, prions, orStaphylococcal enterotoxin B toxicosis.
 6. The method of claim 1 whereinthe extracellular matrix comprises small intestinal submucosa (SIS). 7.The method of claim 1 wherein the extracellular matrix comprising renalcapsule tissue.
 8. The method of claim 1 wherein the extracellularmatrix comprises fascial extracellular matrix.
 9. The method of claim 1wherein the antigen preparation of interest comprises an inactivatedpreparation of the antigen of interest.
 10. The method of claim 1wherein the extracellular matrix comprises porcine small intestinalsubmucosa.
 11. The method of claim 1 wherein the adjuvant comprises a1:10 ratio of the extracellular matrix and a pharmaceutically acceptablecarrier solution.
 12. The method of claim 11 wherein thepharmaceutically acceptable carrier solution is sterile saline.
 13. Themethod of claim 1 wherein the animal is immunized by oral administrationor injection of the composition.
 14. The method of claim 1 wherein thecomposition is administered by intramuscular injection or bysubcutaneous injection.
 15. A method for protecting an animal againstinfection from an infectious pathogen of interest comprising: preparinga composition comprising an antigen or toxin from an infectious pathogenof interest and an extracellular matrix, wherein said composition isessentially free of alum; and administering the composition to an animalto provide a treated animal, wherein survival rate to a lethal dose ofthe infectious pathogen of interest is essentially the same in thetreated animal and an animal receiving a composition consisting of theantigen or toxin from an infectious pathogen of interest with alum. 16.The method of claim 15 wherein the toxin is tetanus toxoid.
 17. Themethod of claim 16 wherein the extracellular matrix comprises smallintestinal submucosal tissue (SIS).
 18. The method of claim 17comprising a particulate small intestinal submucosal tissue (SIS). 19.The method of claim 15 wherein the animal is a human.
 20. The method ofclaim wherein the composition comprises a pharmaceutically acceptablecarrier solution.